Measuring degree of polymerisation for meningococcal capsular saccharides that contain sialic acid

ABSTRACT

The degree of polymerisation (DP) is an important parameter for analysis of saccharide antigens, particularly in glycoconjugates. The invention provides methods that can be used to measure DP for capsular saccharides, particularly for meningococcal saccharides e.g. from serogroups W135 and Y. A preferred method is based on reduction of terminal sialic acid residues on saccharides, with DP then being calculated by comparing the molar ratio of total sialic acid to reduced sialic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/597,451, which is the National Stage of International Application No.PCT/IB2005/02264, filed May 23, 2005, which claims the benefit of GBApplication Ser. No. 0411387.4, filed May 21, 2004, each of which ishereby incorporated by reference in its entirety.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention is in the field of analysis and quality control ofvaccines that include bacterial capsular saccharides, and in particularthose where the saccharides are conjugated to a carrier.

BACKGROUND ART

Immunogens comprising capsular saccharide antigens conjugated to carrierproteins are well known in the art. Conjugation converts T-independentantigens into T-dependent antigens, thereby enhancing memory responsesand allowing protective immunity to develop, and the prototype conjugatevaccine was for Haemophilus influenzae type b (Hib) [e.g see chapter 14of ref. 1]. Since the Hib vaccine, conjugated saccharide vaccines forprotecting against Neisseria meningitidis (meningococcus) and againstStreptococcus pneumoniae (pneumococcus) have been developed. Otherorganisms where conjugate vaccines are of interest are Streptococcusagalactiae (group B streptococcus) [2], Pseudomonas aeruginosa [3] andStaphylococcus aureus [4].

Rather than use full-length capsular saccharides, it is possible toselect oligosaccharide fragments of desired size after a hydrolysis step[e.g. ref. 5], and it has been reported that conjugates made withintermediate chain-length oligosaccharides offer improved immunogenicity[e.g. refs. 6 & 7]. Of the three N. meningitidis serogroup C conjugatedvaccines that have been approved for human use, Menjugate™ [8] andMeningitec™ are based on oligosaccharides, whereas NeisVac-C™ usesfull-length polysaccharide. Measurement of oligosaccharide length (e.g.by measuring the degree of polymerisation, or ‘DP’ i.e. the number ofrepeating units in the chain) can therefore be used for indirectassessment of immunogenicity.

Where oligosaccharide fragments are included in a vaccine, qualitycontrol for manufacturing and release requires that oligosaccharideshave a defined length, and that this length is consistent betweenbatches. Thus DP is also useful in quality control, and the EuropeanDirectorate for Quality of Medicines (EDQM) has an Official ControlAuthority Batch Release (OCABR) for conjugated Hib vaccines thatspecifically requires submission of data relating to DP and molecularsize distribution of saccharides used during manufacture. DP can also beused for monitoring vaccine stability. Saccharide antigens can readilydepolymerise at ambient temperatures [9,10], causing a decrease inimmunogenicity and an increase in vaccine heterogeneity. Such changescan be monitored by following DP over time during storage.

Average DP in an oligosaccharide pool can be measured using a number ofmethodologies, and in some cases the choice of method will depend on thesaccharide under analysis. Techniques such as colorimetric and/orenzymatic analysis have been described for oligosaccharides from Hib andfrom serogroups A and C of meningococcus [5,11,12], but the inventorshave found that the glycosidic linkages in the saccharides ofmeningococcal serogroups W135 and Y (‘MenW135’ and ‘MenY’) mean thatthese techniques cannot be used.

Although methods for measuring DP of MenA and MenC saccharides forconjugate vaccines have previously been described [e.g. refs. 5 & 13],there remains a need for methods that can be applied to the saccharidesof serogroups W135 and Y. It is thus an object of the invention toprovide improvements in methods for DP assessment of saccharides, and inparticular to provide methods that can be used to measure DP forsaccharides from meningococcal serogroups W135 and Y.

DISCLOSURE OF THE INVENTION

The inventors have discovered a method that can be used to measure DPfor the capsular saccharides of meningococcal serogroups W135 and Y.Thus the invention provides a process for measuring the degree ofpolymerisation of a capsular saccharide, characterised in that thesaccharide is from meningococcal serogroup W135 or serogroup Y. Themethod is conveniently performed by including a step of chromatographicseparation. In a composition comprising a population of different-sizedcapsular saccharides, the invention provides a process for measuringaverage DP.

The process typically involves hydrolysis of the saccharides to releaseconstituent monosaccharides, with analysis being based on themonosaccharides. Thus the invention provides a process for measuring thedegree of polymerisation of a capsular saccharide from meningococcalserogroup W135 or serogroup Y, wherein the process comprises the stepsof: (i) hydrolysing the saccharide to give a saccharide hydrolysatecontaining monosaccharide subunits; (ii) quantifying the monosaccharidesubunits in the hydrolysate, wherein the quantitative results of step(ii) are used to calculate the degree of polymerisation.

Prior to hydrolysis, the process typically involves chemicalmodification of a terminal residue (either at the reducing terminus orthe non-reducing terminus) of the saccharide such that, after hydrolysisto monosaccharides, the terminal residue can be distinguished fromnon-terminal residues. Thus the invention provides a process formeasuring the degree of polymerisation of a capsular saccharide frommeningococcal serogroup W135 or serogroup Y, wherein the processcomprises the steps of: (i) modifying a terminal monosaccharide subunitof the saccharide, to give a modified terminal monosaccharide; (ii)hydrolysing the saccharide to give a saccharide hydrolysate containingmonosaccharide subunits, including the modified terminal monosaccharide;(iii) quantifying the monosaccharide subunits from the hydrolysate; (iv)quantifying the modified terminal monosaccharide from the hydrolysate;and (v) using the quantitative results of steps (iii) and (iv) tocalculate the degree of polymerisation.

The method is applicable more generally to any saccharide that containsmore than one different type of monosaccharide subunit and includes aterminal sialic acid residue. Advantageously, the method provides DPinformation based only on quantification of the sialic acid residues ina saccharide, without requiring analysis of any other type ofmonosaccharide. Thus the invention provides a process for measuring thedegree of polymerisation of a capsular saccharide, wherein: (a) thesaccharide comprises sialic acid monosaccharide subunits and non-sialicacid monosaccharide subunits; (b) the saccharide has a terminal sialicacid monosaccharide subunit; and (c) the process comprises the steps of(i) modifying a terminal sialic acid subunit of the saccharide, to givea modified terminal sialic acid subunit; (ii) hydrolysing the saccharideto give a saccharide hydrolysate containing sialic acid subunits,including modified terminal sialic acid subunits; (iii) quantifying thesialic acid subunits from the hydrolysate, including the modifiedterminal sialic acid subunits; and (iv) using the quantitative resultsof step (iii) to calculate the degree of polymerisation.

A preferred method of the invention is based on reduction of terminalsialic acid residues on saccharides, with DP then being calculated bycomparing the molar ratio of total sialic acid to reduced sialic acid.The invention thus provides a process for measuring the degree ofpolymerisation of a capsular saccharide, wherein: (a) the saccharidecomprises sialic acid monosaccharide subunits and non-sialic acidmonosaccharide subunits; (b) the saccharide has a terminal sialic acidmonosaccharide subunit; and (c) the process comprises the steps of: (i)reducing the terminal sialic acid monosaccharide subunit to give areduced sialic acid monosaccharide subunit; (ii) hydrolysing thesaccharide to give a hydrolysate containing monosaccharide subunits;(iii) determining the ratio of total (i.e. reduced and non-reduced)sialic acid to reduced sialic acid in the hydrolysate.

In terms of a composition comprising a population of different-sizedcapsular saccharides, the invention provides a process for measuring theaverage degree of polymerisation of the saccharides, wherein: (a) thesaccharides comprise sialic acid monosaccharide subunits and non-sialicacid monosaccharide subunits; (b) the saccharides have terminal sialicacid monosaccharide subunits; and (c) the process comprises the stepsof: (i) reducing the terminal sialic acid monosaccharide subunits togive reduced sialic acid monosaccharide subunits; (ii) hydrolysing thesaccharides to give a saccharide hydrolysate containing monosaccharidesubunits; (iii) determining the ratio of total (i.e. reduced andnon-reduced) sialic acid to reduced sialic acid in the hydrolysate. Thecomposition may include saccharides that do not have terminal sialicacid monosaccharide subunits.

A method for analysing the length and composition of poly-sialic acidsaccharides has been described [14] in which terminal residues areoxidised and/or reduced, after which the saccharide is digested withneuraminidase enzyme to release its sialic acid monosaccharide subunits.However, this prior art process was described only for saccharidescomposed solely of sialic acids (including N-acetyl-neuraminic acid,N-glycolyl-neuraminic acid and deaminated sialic acid) and wastechnically limited to saccharides that can be enzymatically cleavedinto monosaccharides. In contrast, the method of the invention can dealwith saccharides that include non-sialic acid monosaccharides, and doesnot require (but does not exclude) the use of enzymatic hydrolysis.

The inventors have also discovered a method that can be used to measureDP for the capsular saccharides of meningococcal serogroup C. The methodis again based on reduction of terminal sialic acid residues, with DPbeing calculated in the same way. The method is applicable moregenerally to any saccharide that contains sialic acid residues that arelinked α 2→9. Preferably, the method does not involve enzymaticdepolymerisation. The invention thus provides a process for measuringthe degree of polymerisation of a capsular saccharide, wherein: (a) thesaccharide comprises sialic acid monosaccharide subunits that are linkedα 2→9; (b) the saccharide has a terminal sialic acid monosaccharidesubunit; and (c) the process comprises the steps of: (i) reducing theterminal sialic acid monosaccharide subunit to give a reduced sialicacid monosaccharide subunit; (ii) hydrolysing the saccharide to give ahydrolysate containing monosaccharide subunits; (iii) determining theratio of total (i.e. reduced and non-reduced) sialic acid to reducedsialic acid in the hydrolysate.

In terms of a composition comprising a population of different-sizedcapsular saccharides, the invention provides a process for measuring theaverage degree of polymerisation of the saccharides, wherein: (a) thesaccharide comprises sialic acid monosaccharide subunits that are linkedα 2→9; (b) the saccharides have terminal sialic acid monosaccharidesubunits; and (c) the process comprises the steps of: (i) reducing theterminal sialic acid monosaccharide subunits to give reduced sialic acidmonosaccharide subunits; (ii) hydrolysing the saccharides to give ahydrolysate containing monosaccharide subunits; (iii) determining theratio of total (i.e. reduced and non-reduced) sialic acid to reducedsialic acid in the hydrolysate.

A method for analysing the length and composition of poly-sialic acidsaccharides has been described [14] in which terminal residues areoxidised and/or reduced, after which the saccharide is digested withneuraminidase enzyme to release its sialic acid monosaccharide subunits.However, this prior art process was described only for saccharidescomposed of α 2→8 linked sialic acids and was technically limited tosaccharides that can be enzymatically cleaved into monosaccharides. Incontrast, the method of the invention is concerned with α 2→9 linkedsialic acids, and preferably utilises non-enzymatic hydrolysis,typically chemical (e.g. acidic) hydrolysis.

A method for determining the length of a serogroup C saccharide is known[5] where DP was determined by comparing the ratio between total sialicacid and formaldehyde generated by periodate treatment of the MenCsaccharide. This prior art method involves modification at thenon-reducing terminus of the polymer, and does not involve thegeneration of sialitol.

Capsular Saccharides Containing Sialic Acid Residues and Non-sialic AcidResidues

In some embodiments, the invention provides methods for analysingsaccharides that comprise both sialic acid monosaccharide subunits andnon-sialic acid monosaccharide subunits. More particularly, thesaccharides are preferably made up of repeating units, and the repeatingunits consist of sialic acid monosaccharide subunits and non-sialic acidmonosaccharide subunits.

Two particular saccharides of interest are the capsular saccharides ofNeisseria meningitidis serogroups W135 and Y. The saccharides naturallyhave a sialic acid residue at their reducing end and either glucose orgalactose at the non-reducing end. The sialic acid in the nativesaccharides of these serogroups is N-acetyl neuraminic acid, or‘NeuNAc’.

The serogroup W135 saccharide is a polymer consisting of sialicacid-galactose disaccharide units. It has variable O-acetylation at the7 and 9 positions of the sialic acid [15]. The structure is shown inFIG. 1 and is written as: →4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→

The serogroup Y saccharide is similar to the serogroup W135 saccharide,except that the disaccharide repeating unit includes glucose instead ofgalactose (see FIG. 3). It has variable O-acetylation at the 7 and 9positions of the sialic acid [15]. The serogroup Y structure is shown inFIG. 2 and is written as: →4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→

In other embodiments, the invention provides methods for analysingsaccharides that comprise (α 2→9)-linked sialic acids. The serogroup Ccapsular saccharide is a homopolymer of (α 2→9) linked sialic acid, withvariable O-acetylation at positions 7 and/or 8.

The capsules of serogroups C, W135 and Y differ from serogroup A, whichhas a homopolymer of (α1→6)-linked N-acetyl-D-mannosamine-1-phosphate,and from serogroup B, which has a homopolymer of (α 2→8)-linked sialicacid.

Degree of Polymerisation

The degree of polymerisation of a saccharide is defined as the number ofrepeating units in that saccharide. For a homopolymer, the degree ofpolymerisation is thus the same as the number of monosaccharide units.For a heteropolymer, however, the degree of polymerisation is the numberof monosaccharide units in the whole chain divided by the number ofmonosaccharide units in the minimum repeating unit e.g. the DP of(Glc-Gal)₁₀ is 10 rather than 20, and the DP of (Glc-Gal-Neu)₁₀ is 10rather than 30.

Within a mixture of saccharides having the same basic repeatingstructure but different lengths (e.g. in a partial hydrolysate of a longpolysaccharide) then it is normal to measure the average DP of apopulation rather than to measure the DP of individual molecules. If thesize range is too large, such that an average value will not bemeaningful, then it is possible to measure DP for individual fractionsof a mixture after separation (e.g. after separation by size). Ingeneral, the invention will be used to measure the average DP ofcompositions containing mixed-length saccharides.

Reduction of the Terminal Sialic Acid Monosaccharide Subunit

The invention is used to analyse saccharides that have terminal sialicacid residues. In particular, it is used to analyse saccharides thathave sialic acid residues at the reducing terminus.

Any suitable chemistry can be used for reduction of the terminal sialicacid residue, generally involving incubating the saccharide with areducing agent. Suitable conditions for any given reducing agent and anygiven saccharide can be determined by routine analysis.

A preferred reducing agent is sodium borohydride (NaBH₄), which reducesterminal sialic acid residues [14] under alkaline conditions. Theproduct of this reduction is sometimes referred to as sialitol [16].Incubation with NaBH₄ for 2 hours at 37° C. is generally adequate. Afterreduction in alkaline conditions then a composition is preferablyneutralised e.g. by adding mildly-acidic ammonium acetate.

Hydrolysing the Saccharide to Give a Saccharide Hydrolysate

After reduction of the terminal sialic acid residue, the saccharide isbroken into its constituent monosaccharide units. In general terms,depolymerisation of saccharides to yield monosaccharides can beperformed chemically or enzymatically. If there is no enzyme forperforming a given cleavage reaction, however, then the chemical routemust be used.

Chemical hydrolysis of saccharides generally involves treatment witheither acid or base under conditions that are standard in the art.Conditions for depolymerisation of capsular saccharides to theirconstituent monosaccharides are known in the art. For serogroup W135 andY saccharides, acid hydrolysis is preferred. Acid hydrolysis using TFA(trifluoroacetic acid) can be used for hydrolysis of all of serogroupsC, W135 and Y, with a slightly lower incubation temperature beingpreferred for serogroup C to avoid degradation of its sialic acids (90°C. rather than 100° C.). A typical TFA treatment involves addition ofTFA to a final concentration of 2 M, followed by heating to 90-100° C.for 90 minutes. The serogroup C saccharide can be hydrolysed for totalsaccharide content analysis by treatment with 100 mM HCl at 80° C. for 2hours [17]. Other typical hydrolysis conditions involve millimolarconcentrations of a weak acid (e.g. acetic acid) at elevatedtemperatures (e.g. 70-80° C.).

Enzymes are available for cleaving the α 2→9 linkages found in serogroupC, and these may be used with the invention. However, enzymes generallyrequire the saccharides to be de-O-acetylated prior to hydrolysis, so ifit is desired to maintain O-acetylation [15] then it is preferred tohydrolyse the saccharide chemically. Chemical hydrolysis may also bepreferred where enzymatic hydrolysis proceeds slowly. Enzymes forcleaving serogroups W135 and Y are not generally available.

Although the invention has been defined above in terms of preparing andanalysing a hydrolysate containing monosaccharide subunits, theinvention can also be applied to hydrolysates containing disaccharide,trisaccharide, tetrasaccharide etc. fragments of the capsularsaccharide, but it is easier to prepare a hydrolysate ofmonosaccharides. Hydrolysis conditions that provide a homogenouspopulation of di-, tri-, tetra- etc. saccharides, such that there isonly a single compound to be quantified, are much more difficult tocontrol than simply allowing depolymerisation to proceed to completioni.e. to give monosaccharides.

After depolymerisation, saccharide hydrolysates may be dried e.g. usinga vacuum drier.

Determining the Ratio of Total Sialic Acid to Reduced Sialic Acid

Hydrolysis gives a saccharide hydrolysate that contains themonosaccharide subunits of the original saccharide. In embodiments wheresaccharides comprise both sialic acid subunits and non-sialic acidsubunits, the hydrolysate will contain sialic acid and non-sialic acidmonosaccharides; in embodiments where saccharides are sialic acidhomopolymer then the hydrolysate will contain only sialic acids; in bothcases, a fraction of the sialic acid monosaccharides will be in amodified form (e.g. a reduced form). That fraction can be used todetermine the DP of the original saccharide. For example, if one in tenof the sialic acid residues in the mixture is a modified residue and theminimum repeating unit of the saccharide contains a single sialic acidresidue then the original saccharide has a DP of 10.

Quantities of individual monosaccharides can be determined in terms ofnumbers (e.g. moles) of molecules, masses, ratios or concentrations. Itis typical to work in moles in order to simplify the calculation ofratios, particularly where constituent monosaccharides have differentmolecular masses, but any of these measures can be used and interchangedto determine monosaccharide content of the mixtures. For quantitativemeasurement, analytical results may be compared to a standard with aknown content of a particular saccharide.

The depolymerised mixture is preferably hydrolysed completely tomonosaccharides. The inventors have found that incomplete hydrolysissometimes occurs, giving mixtures in which disaccharide fragments arepresent (i.e. Gal-NeuNAc for MenW135, and Glc-NeuNAc for MenY). Forinstance, treatment of MenW135 or MenY saccharides with 2M TFA at 90° C.has been seen to give a mixture of monosaccharides and disaccharides,whereas increasing the hydrolysis temperature to 100° C. givessubstantially only monosaccharides. Incomplete hydrolysis even at 90° C.was not expected but, now that it has been observed, the skilled personcan, if necessary, modify any particular hydrolysis method to ensuretotal hydrolysis e.g. by increasing temperature, etc.

Methods for quantifying sialic acid monosaccharides are well known inthe art. Methods may be direct or indirect (e.g. they may involvederivatisation of the monosaccharides followed by an analysis thatcorrelates with original monosaccharide content). Preferred methods cananalyse sialic acid in the presence of other monosaccharides, such thatthey do not need to be separated from each other before analysis. Inaddition, methods may be used for conjugated saccharides in which, afterdeconjugation, the carrier and the saccharide need not be separated. Onesuch method is anion chromatography, and in particular high performanceanion exchange chromatography (HPAEC), usually with pulsed amperometricdetection (PAD) [18,19]. HPAEC-PAD systems are provided by Dionex™Corporation (Sunnyvale, Calif.) e.g. the BioLC™ system, using a columnsuch as PA1 [10 μm diameter polystyrene substrate 2% crosslinked withdivinylbenzene, agglomerated with 500 nm MicroBead quaternary ammoniumfunctionalized latex (5% crosslinked)] or PA10 [10 μm diameterethylvinylbenzene substrate 55% crosslinked with divinylbenzene,agglomerated with 460 nm MicroBead difunctional quaternary ammonium ion(5% crosslinked)]. These systems can quantitatively analyse individualsaccharides within mixtures without the need for derivatisation orpre-analysis separation. For saccharide analysis, it may be desired tofilter other compounds before entry to the column, and Dionex™ producepre-column traps and guards for this purpose e.g. an amino trap forremoving amino acids, a borate trap, etc.

An alternative method for quantifying sialic acid monosaccharides withina depolymerised mixture is nuclear magnetic resonance (NMR). For ease ofuse and for high sensitivity, however, the chromatographic methods ofthe invention are preferred. Whichever method is chosen, however, insome embodiments of the invention it is important that reduced sialicacid can be distinguished from non-reduced sialic acid. This may involveunique signals from each, or may involve one unique signal and onecombined signal, with the difference between the two giving signalsproviding the necessary information.

Another method for quantifying sialic acid monosaccharides is bycolorimetric assay [80]. This method is particularly useful forquantifying non-reduced sialic acid after acid hydrolysis in TFA.

Once the relative quantities of modified and non-modified (e.g. reducedand non-reduced) sialic acid have been determined then it is simple toestablish the DP of the original saccharide.

In addition to quantifying sialic acids in the hydrolysate, methods ofthe invention may involve quantification of other monosaccharides (e.g.of glucose or galactose) which may be derived from the same saccharideas the sialic acids, or from other saccharides. These measurements canbe used for determining parameters other than DP, or can be used as partof the DP determination e.g. as confirmation or in place of measurementof total sialic acid, particularly where the molar quantities of sialicacid and the other monosaccharide are the same, as in the W135 and Ysaccharides.

The process of the invention is typically destructive. Rather thanperform the process on a complete composition, therefore, it is moretypical to take a sample from a composition of interest and then performthe analysis on the sample.

Conjugates

The invention is useful for analysing saccharide content of vaccines,and in particular for vaccines that comprise a conjugated saccharide.Covalent conjugation is used to enhance immunogenicity of saccharides byconverting them from T-independent antigens to T-dependent antigens,thus allowing priming for immunological memory. Conjugation isparticularly useful for paediatric vaccines and is a well knowntechnique [e.g. reviewed in refs. 20 to 29]. Saccharides may be linkedto carriers directly [30, 31], but a linker or spacer is generally usede.g. adipic acid, β-propionamido [32], nitrophenyl-ethylamine [33],haloacyl halides [34], glycosidic linkages [35], 6-aminocaproic acid[36], ADH [37], C₄ to C₁₂ moieties [38], etc.

Typical carrier proteins in conjugates are bacterial toxins or toxoids,such as diphtheria toxoid or tetanus toxoid. The CRM₁₉₇ diphtheria toxinderivative [39-41] is the carrier protein in Menjugate™ and Meningitec™,whereas tetanus toxoid is used in NeisVac™. Diphtheria toxoid is used asthe carrier in Menactra™. Other known carrier proteins include the N.meningitidis outer membrane protein [42], synthetic peptides [43,44],heat shock proteins [45,46], pertussis proteins [47,48], cytokines [49],lymphokines [49], hormones [49], growth factors [49], artificialproteins comprising multiple human CD4⁺ T cell epitopes from variouspathogen-derived antigens [50], protein D from H. influenzae [51,52],pneumococcal surface protein PspA [53], iron-uptake proteins [54], toxinA or B from C. difficile [55], etc. Compositions may use more than onecarrier protein e.g. to reduce the risk of carrier suppression, and asingle carrier protein might carry more than one saccharide antigen[56]. Conjugates generally have a saccharide:protein ratio (w/w) ofbetween 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide).Compositions may include free carrier protein in addition to theconjugates [57].

The invention is particularly useful prior to conjugation at the stagewhere it is necessary to ensure that the correctly-sized saccharidechains are selected for production of the conjugate.

The invention allows the progress of fragmentation of a full-lengthpolysaccharide prior to conjugation to be checked or monitored. Whereoligosaccharides of a particular length (or range of lengths) is desiredthen it is important that fragmentation of the polysaccharide should notbe so extensive as to take depolymerisation past the desired point (e.g.at the extreme, to give monosaccharides). The invention allows theprogress of this partial depolymerisation to be monitored, by measuringaverage chain length over time. Thus the invention provides a processfor measuring the degree of polymerisation of saccharide(s) in acomposition, comprising the steps of: (a) starting depolymerisation ofthe saccharide(s) in the composition; and, at one or more time pointsthereafter, (b) measuring DP of the saccharide(s) as described above. Inan initial run of experiments then it will be usual to measure DP atseveral time points in order to determine progress over time, but afterstandard conditions have been established then it be usual to measure DPat a set time point for confirmatory purposes. Once DP is at a desiredlevel then the process may comprise the further step of: (c) stoppingthe depolymerisation, e.g. by washing, separating, cooling, etc. Theprocess may also comprise the further step of conjugation of thedepolymerised saccharide to a carrier protein, after optional chemicalactivation.

The invention also allows selection of oligosaccharide chains of adesired length after fragmentation. Thus the invention provides aprocess for selecting saccharides for use in preparing a glycoconjugate,comprising the steps of: (a) obtaining a composition comprising amixture of different polysaccharide fragments having different degreesof polymerisation; (b) separating the mixture into sub-mixtures; (c)determining the DP of one or more sub-mixtures using a process asdescribed above; and (d) using the results of step (c) to select one ormore sub-mixtures for use in conjugation. The process may involvefragmentation of the polysaccharide prior to step (a), or may start withan already-prepared mixture. The fragments may be fragments of the samepolysaccharide e.g. of the same serogroup. After step (d), the processmay comprise the step of conjugation to a carrier protein, afteroptional chemical activation.

Prior to conjugation it is usual for a saccharide to be chemicallyactivated in order to introduce a functional group that can react withthe carrier. Conditions for saccharide activation can cause hydrolysis,and so it is useful to check DP after activation. The term “saccharide”should, where appropriate, be taken to include these activatedsaccharides. Moreover, the invention provides a process for preparing anactivated saccharide for use in preparing a glycoconjugate, comprisingthe steps of: (a) obtaining a saccharide; (b) chemically activating thesaccharide to introduce a functional group that can react with a carrierprotein; and (c) measuring the DP of the product of step (b) asdescribed above. The process may include the further step of: (d)reacting the activated saccharide with the carrier protein (which mayitself have been activated) to give the glycoconjugate. The process mayinvolve fragmentation of a polysaccharide prior to step (a), or maystart with an already-prepared mixture.

The invention can also be used after conjugation. After conjugation,compositions can be analysed using the invention in three ways: first,the DP of total saccharides in a composition can be measured e.g. priorto mixing of different conjugates, or prior to release of a vaccine (forregulatory or quality control purposes); second, the DP of freeunconjugated saccharide in a composition can be measured e.g. to checkfor incomplete conjugation, or to follow conjugate hydrolysis bymonitoring increasing free saccharide over time; third, the DP ofconjugated saccharide in a composition can be measured, for the samereasons. The first and third ways require the saccharide to be releasedfrom the conjugate prior to analysis. In situations where conjugation ofthe saccharide involved reaction or modification of the sialic acidresidue its reducing end, however, such that the residue is no longeramenable to reduction, then the invention can be used only forsaccharides where a terminal sialic acid can be re-generated (or where areduced terminal sialic acid can be generated directly).

To separately assess conjugated and unconjugated saccharides, they mustbe separated. Free (i.e. unconjugated) saccharide in an aqueouscomposition can be separated from conjugated saccharide in various ways.The conjugation reaction changes carious chemical and physicalparameters for the saccharide, and the differences can be exploited forseparation. For example, size separation can be used to separate freeand conjugated saccharide, as the conjugated material has a higher massdue to the carrier protein. Ultrafiltration is a preferred sizeseparation method. As a further alternative, if conjugates have beenadsorbed to an adjuvant then centrifugation will separate adsorbedconjugate (pellet) from free saccharide (supernatant) that desorbs afterhydrolysis.

The invention provides a method for analysing a glycoconjugate,comprising the steps of: (a) treating the glycoconjugate to releasesaccharide from carrier; and (b) measuring DP of the released saccharideas described above. The invention provides a method for analysing aglycoconjugate composition, comprising the steps of: (a) separatingunconjugated saccharide within the composition from conjugatedsaccharide; and (b) measuring DP of the unconjugated and/or conjugatedsaccharide as described above.

The invention also provides a method for releasing a vaccine for use byphysicians, comprising the steps of: (a) manufacturing a vaccinecomprising a conjugate of a capsular saccharide, wherein the saccharidecomprises sialic acid monosaccharide subunits and non-sialic acidmonosaccharide subunits; (b) analysing DP of saccharide in the vaccineas described above; and, if the results from step (b) indicate a DPacceptable for clinical use, (c) releasing the vaccine for use byphysicians. Step (b) may be performed on a packaged vaccine or on a bulkvaccine prior to packaging.

Mixed Saccharides

The invention allows DP analysis in compositions that comprisemeningococcal capsular saccharides that include sialic acid. Thecompositions may also comprise further capsular saccharides (e.g. acapsular saccharide from serogroup A of N. meningitidis, a capsularsaccharide from H. influenzae b, etc.) provided that these saccharidesdo not contain sialic acids, which would interfere with the overallanalysis. Where more than one saccharide in a composition includessialic acid residues then the principles disclosed in reference 58 canbe used to distinguish the different saccharides.

The capsular saccharide of serogroup A meningococcus is a homopolymer of(α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, with partialO-acetylation in the C3 and C4 positions. The acetyl groups can bereplaced with blocking groups to prevent hydrolysis [10], and suchmodified saccharides are still serogroup A saccharides within themeaning of the present invention.

The Hib capsular saccharide is a polymer of ribose, ribitol, andphosphate. The saccharide is known as ‘PRP’(poly-3-β-D-ribose-(1,1)-D-ribitol-5-phosphate).

Saccharide Components Other than Capsular Saccharides

It is preferred that compositions for analysis by the invention do notinclude sialic acid in free form (other than any backgroundmonosaccharides derived from capsular saccharide hydrolysis). If freesialic acid is present, however, then there are two general ways inwhich interference problems can be minimised or avoided. First, initiallevels of free sialic acid can be measured and then subtracted from thelevels measured in the depolymerised mixture. Second, free sialic acidcan be removed from the composition prior to analysis e.g. by filtrationor dialysis. Ultrafiltration membranes can be used to remove lowmolecular weight components.

Non-saccharide Components

As well as analysing saccharides in a composition, the process mayinclude analysis of other components or properties e.g. osmolality, pH,degree of polymerisation for individual saccharides or conjugates,protein content (particularly for carrier proteins), aluminium content,detergent content, preservative content, etc.

The invention provides a method for preparing a vaccine composition,comprising a step of DP analysis of a saccharide according to theinvention, and a step of pH measurement of the composition, optionallyfollowed by a step of adjusting the pH of the composition to a desiredvalue e.g. between 6 and 8, or about 7.

The invention also provides a method for preparing a vaccinecomposition, comprising the steps of: (a) providing DP-analysed capsularsaccharide as described above; (b) conjugating the DP-analysedsaccharide to one or more carrier proteins; (c) optionally, analysingthe bulk vaccine for pH and/or other properties; and, if the resultsfrom step (c) indicate that the bulk vaccine is acceptable for clinicaluse, (d) preparing and packaging the vaccine for human use from thebulk. Step (c) may involve assessment of minimum saccharideconcentration, assessment of unconjugated:conjugated saccharide ratio,etc. Step (d) may involve packaging into unit dose form or in multipledose form e.g. into vials or into syringes. A typical human dose forinjection has a volume of 0.5 ml.

The invention also provides a method for preparing a vaccinecomposition, comprising the steps of: (a) providing DP-analysed capsularsaccharide from serogroup W135 and/or Y, as described above; (b)conjugating the DP-analysed saccharide to one or more carrier proteins,to give conjugated saccharide; and (c) mixing the conjugated saccharidewith one or more further antigens e.g. with

-   -   a capsular saccharide antigen from serogroup C of N.        meningitidis.    -   a capsular saccharide antigen from serogroup A of N.        meningitidis.    -   a protein antigen from serogroup B of N. meningitidis.    -   preparations of N. meningitidis serogroup B microvesicles [59],        ‘native OMVs’ [60], blebs or outer membrane vesicles [e.g. refs.        61 to 66 etc.].    -   a saccharide antigen from Haemophilus influenzae type b.    -   an antigen from Streptococcus pneumoniae, such as polyvalent        conjugated saccharide antigens [e.g. refs. 67 to 69].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 70, 71].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 71, 72].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 73 & 74]. Cellular pertussis        antigens may be used.    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 75] e.g. the CRM₁₉₇ mutant [e.g. 76].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 75].    -   polio antigen(s) [e.g. 77, 78], such as IPV.

Such antigens may be adsorbed to an aluminium salt adjuvant (e.g. ahydroxide or a phosphate). Any further saccharide antigens arepreferably included as conjugates.

Batch-to-Batch Consistency

For human vaccine manufacture, conjugated saccharides should besubjected to quality control before conjugation (e.g. the saccharide andthe carrier protein), after conjugation, after formulation and aftermixing. Prior art methods for DP measurement do not relate to thesaccharides from serogroups W135 and Y. With the invention, however, DPmeasurement for these two serogroups is now possible, and can becombined with methods for DP measurement of serogroups A and C [5].Moreover, the processes of the invention are reliable and consistent,and thus allow valid comparisons of different batches of mixedA/C/W135/Y conjugates, where this was not possible with prior artmethods. Different batches of mixed conjugate vaccines can thus beprepared, assayed, and consistent batches can be selected for releaseand use, whereas aberrant batches can be rejected.

The invention provides two batches of a vaccine, wherein: (a) both ofthe batches of vaccine comprise: (i) a conjugate of a capsularsaccharide from serogroup A of Neisseria meningitidis; (ii) a conjugateof a capsular saccharide from serogroup C of Neisseria meningitidis;(iii) a conjugate of a capsular saccharide from serogroup W135 ofNeisseria meningitidis; (iv) a conjugate of a capsular saccharide fromserogroup Y of Neisseria meningitidis; (b) the DP of the serogroup Asaccharide in the first batch is A₁ and the DP of the serogroup Asaccharide in the second batch is A₂; (c) the DP of the serogroup Csaccharide in the first batch is C₁ and the DP of the serogroup Csaccharide in the second batch is C₂; (d) the DP of the serogroup W135saccharide in the first batch is W₁ and the DP of the serogroup W135saccharide in the second batch is W₂; (e) the DP of the serogroup Ysaccharide in the first batch is Y₁ and the DP of the serogroup Ysaccharide in the second batch is Y₂; (f) the ratios A₁/A₂, C₁/C₂, W₁/W₂and Y₁/Y₂ are each between 0.90 and 1.10, and preferably are eachbetween 0.95 and 1.05.

The ratios specified in (f) may be based on a single sample from eachbatch being compared, but will typically be based on average values(e.g. means) from multiple samples of each batch. Thus the two batchesmay be subjected to multiple sampling, and each sample may be subjectedto multiple measurements of A₁, A₂, C₁, C₂, W₁, W₂, Y₁ and Y₂, withaverages then being calculated for each batch, and with the averagesbeing used to calculate the necessary ratios.

Each batch (or lot) of vaccine will have been prepared separately. Forexample, two different batches can be made by separate mixings of thesame bulk single conjugates, or by mixing bulk single conjugates thatwere separately prepared. Different samples of the same bulk mixture arenot different batches, as these samples are not subject to thebatch-to-batch variations that result from differences that arise whenpreparing mixtures of different conjugates.

In addition to characteristics (a) to (f) as specified above, the twobatches may additionally be characterised by: (g) the concentration ofunconjugated serogroup A saccharide in the first batch is A₃; (h) theconcentration of unconjugated serogroup A saccharide in the second batchis A₄; (i) the concentration of unconjugated serogroup C saccharide inthe first batch is C₃; (j) the concentration of unconjugated serogroup Csaccharide in the second batch is C₄; (k) the concentration ofunconjugated serogroup W135 saccharide in the first batch is W₃; ifapplicable, (l) the concentration of unconjugated serogroup W135saccharide in the second batch is W₄; (m) the concentration ofunconjugated serogroup Y saccharide in the first batch is Y₃; (n) theconcentration of unconjugated serogroup Y saccharide in the second batchis Y₄; (o) the ratios A₃/A₄, C₃/C₄, W₃/W₄ and Y₃/Y₄ are each between0.90 and 1.10, and preferably are each between 0.95 and 1.05.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x+10%.

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 show structural formulae for the capsular saccharides ofmeningococcal serogroups W135 (11), and Y (2).

FIG. 3 shows the difference between serogroups W135 and Y.

FIGS. 4 and 5 show chromatograms of MenY saccharides before (4) andafter (5) size separation.

FIGS. 6 and 7 show ESI spectra for MenW135 (6) and MenY (7) saccharides.

FIG. 8 shows the structure of the MenW135 DP3 saccharide above its ¹HNMR spectrum.

FIG. 9 shows the same for MenY DP4 saccharide.

FIGS. 10 to 12 show isocratic elution profiles of sialic acid standardsolutions.

FIG. 13 shows a gradient elution profile of MenY oligosaccharide.

FIG. 14 shows a standard curve for sialitol.

FIG. 15 shows the decrease in DP of MenW135 (▭) and MenY (⋄) duringdepolymerisation as measured by HPAEC-PAD. FIGS. 16 and 17 show theincrease in short length oligos during the same process, for MenW135(16) and MenY (17), comparing time zero (A) with final mixtures (B).

FIG. 18 shows an ESI spectrum of MenC DP5, and

FIG. 19 shows HPAEC analysis of the same.

FIG. 20 is a gradient elution profile of MenW135 oligosaccharide. Theleft axis shows amperometric detection in nC; the right axis shows %eluent (100 mM Na-acetate+20 mM NaOH).

FIG. 21 is a further gradient elution profile of MenW135oligosaccharide. FIG. 21A shows a standard sample, and FIG. 21B showsthe MenW135 material.

MODES FOR CARRYING OUT THE INVENTION

Preparation of Standard Oligosaccharide Solutions

Purified serogroup W135 and Y capsular polysaccharide (CPS) wereprepared using the methods described in reference 79. They was suppliedas a 10 mg/ml solution in 0.01 M acetic acid. To hydrolyse the CPS, theywere heated to 70-80° C. for a prolonged period. During the hydrolysis,samples were obtained from the solutions for analysis, and were cooledand neutralized after being extracted. Fragments resulting from thishydrolysis have terminal sialic acid residues, rather than terminalglucose or galactose residues. Oligosaccharides were then purified byion exchange chromatography on a Q-Sepharose column, which separates onthe basis of size and charge. For initial normalisation, sialic acidcontent was measured by a modified Svennerholm method [80], whereby theabsorbance was read at 564 nm. Specific fractions were isolated andanalysed by NMR, by electrospray mass spectrometry and by HPAEC.

HPAEC analysis of the oligosaccharides used either a CarpoPac PA100 oran IonPac AS11 column (both 4×250 mm) on a Dionex DX-500 chromatographysystem fitted with a GP40 pump, ED40 detector and AS3500 auto sampler.Separations were performed at room temperature using a flow rate of 1.0ml/min.

The PA100 column used the following eluents: A) sodium acetate 500mM+sodium hydroxide 100 mM and B) sodium hydroxide 100 mM. An initialisocratic elution with 10% A (15 min) was followed by a linear gradientelution from 10% to 100% A over 50 min. The AS11 column used thefollowing eluents: A) sodium hydroxide 100 mM and B) water, with alinear gradient elution from 5% to 100% A over 50 min.

Eluates were monitored using a pulsed electrochemical detector (ED40) inthe pulsed amperometric mode with a gold working electrode and anAg/AgCl reference electrode. A triple-potential waveform was appliedusing the following settings: E1=0.05 V, t1=400 ms; E2=0.75 V, t2=200ms; E3=0.15 V, t3-400 ms. Integration occurs from 200 ms to 400 msduring E1 application. The resulting chromatographic data wereintegrated and processed using Peak Net data reduction software(Dionex).

The CarboPac PA100 HPAEC gave a profile of MenW135 and MenYoligosaccharides. Calibration of the spectra was performed by comparingpurified and pool oligosaccharide chromatograms, with parallelcharacterisation of purified oligosaccharides by ESI-MS to allowcorrelation between the peak number in the chromatogram and the DP ofthe eluting oligosaccharides.

ESI mass spectrometry was performed on a Micromass ZQ-4000 mass analyserequipped with an electrospray ionization source. The instrument wascalibrated using a sodium iodide (2 g/l) and caesium iodide (50 mg/l)diluted with isopropyl alcohol/water 50/50 (v/v). The capillary voltagewas 3 kV, the cone voltage was 60 V. Samples were dissolved in 50% (v/v)aqueous acetonitrile+0.1% formic acid and injected with flow rate of 10μl/min. The spectra were recorded in positive ion mode with scanningrange from 200 to 2000 m/z.

FIGS. 4 and 5 show HPAEC chromatograms (PA100 column) for the MenYoligosaccharide mixture before (FIG. 4) and after (FIG. 5) Q-Sepharoseseparation. ESI analysis of the peak at ˜38 minutes confirmed that it isa MenY oligosaccharide with a DP of 4 (FIG. 7). Similar experiments wereperformed for MenW135, with FIG. 6 showing an ESI spectrum of a DP3oligosaccharide.

In the ESI analysis, a single molecular species can show multiplemolecular ions in the spectrum, corresponding to the parent moleculewith varying numbers of O-acetyl substitution and sodium ions adduct.Sodium adduct ions can arise from the presence of some amounts of sodiumduring sample analysis and are commonly observed in the mass spectra ofoligosaccharides. Furthermore the parent molecule can assume differentnumber of positive charges, and so an oligosaccharide molecule canproduce many different positive ions, depending on O-acetylsubstitution, sodium adducts and number of positive charge. There arethus large numbers of positive ions in FIGS. 6 and 7.

In FIG. 6, the peaks between 700-800 and 1400-1526 m/z correspond todouble and single-charged ions respectively, resulting from the additionof sodium cations and O-Acetyl groups. They were assigned to thefollowing monoisotopic masses, which correspond to those of a trimeroligosaccharide (MenW135 DP3):

Observed ions Expected ions^(a) O-acetyl^(b) Na^(c) charge 1400.6 1401.40 1 1 1442.8 1443.4 1 1 1 1484.8 1485.4 2 1 1 1526.5 1527.4 3 1 1 712.0712.7 0 2 2 723.0 1 2 733.1 733.7 1 2 2 744.2 2 754.0 754.7 2 2 2 765.13 2 775.6 775.7 3 2 2 ^(a)theoretical ions calculated from monoisotopicmasses by MW calculator (MassLynx software) ^(b)number of O-acetylsubstituents ^(c)number of sodium as counter ions

In FIG. 7, the peaks between 900-1100 and 1800-2000 m/z correspond todouble and single-charged ions respectively, resulting from the additionof sodium cations and O-Acetyl groups. They were assigned to thefollowing monoisotopic masses showed in Tab. 2, which corresponds tothose of a tetramer oligosaccharide (MenY DP4):

Observed ions Expected ions O-acetyl Na charge 1896.0 1896.6 1 1 1 916.80 0 2 927.7 927.8 0 1 2 938.7 937.8 1 0 2 949.6 949.3 1 1 2 959.7 958.82 0 2 971.4 970.3 2 1 2 980.8 979.8 3 0 2 991.7 991.3 3 1 2 1003.31002.8 3 2 2

NMR samples were prepared by dissolving lyophilised oligosaccharides in750 μL 99.9% D₂O (Aldrich™) to give 10-15 mM concentrated solutions. 5mm Wilmad™ NMR tubes were used for every experiment. NMR spectra wererecorded at 298 K on a Bruker™ NMR Spectrometer Avance DRX 600 MHz,equipped with a 5 mm TBI triple resonance heteronuclear probe and a BGUunit. Bruker XWINNMR 3.0 software was used for data acquisition andprocessing. ¹H standard spectral acquisition conditions were to collect32 k data points over a spectral window of 6000 Hz with 4 scans and 10sec of relaxation delay. ¹H NMR spectra were Fourier-transformed afterapplying a 0.1 Hz line broadening function and referenced relative tomono-deuterated water at 4.79 ppm. ¹³C and 2D NMR experiments(double-quantum filtered COSY and HSQC) were carried out to assign the¹H spectra of oligosaccharides.

Proton NMR spectra of the meningococcal W135 and Y oligosaccharides wereassigned by comparison with published data [81] and by two-dimensionalproton-proton COSY and proton-carbon HSQC correlation spectra. Inaddition to proton chemical shifts, the homonuclear and heteronuclearcoupling constants offered a wealth of structural information. Thehigh-resolution NMR spectra of W135 (FIG. 8) and MenY (FIG. 9)oligosaccharides show relatively sharp signals which allow aparticularly defined peak assignment of anomeric proton of Gal/Glcmoieties and the H3 eq/axNeuNAc of NeuNAc moieties. These peaks aresituated in spectral regions where there is no superposition with othersignals that can complicate linear resolution. By expanding thespectrum, proton chemical shifts of signals could be determined:

W135 ppm Y ppm H₁ ^(Gal) _(UR1) 5.112 H₁ ^(Glc) _(UR1) 5.090 H₁ ^(Gal)_(UR2) 5.095 H₁ ^(Glc) _(UR2) 5.049 H₁ ^(Gal) _(UR3) 5.087 H₁ ^(Glc)_(UR3) 5.049 H_(3eq) ^(NeuNAc) _(UR3) 2.963 H₁ ^(Glc) _(UR4) 5.025H_(3eq) ^(NeuNAc) _(UR2) 2.905 H_(3eq) ^(NeuNAc) _(UR4) 2.918 H_(3eq)^(NeuNAc) _(UR1) 2.434 H_(3eq) ^(NeuNAc) _(UR3) 2.907 H_(3ax) ^(NeuNAc)_(UR1) 1.762 H_(3eq) ^(NeuNAc) _(UR2) 2.897 H_(3ax) ^(NeuNAc) _(UR2)1.707 H_(3eq) ^(NeuNAc) _(UR1) 2.400 H_(3ax) ^(NeuNAc) _(UR3) 1.687H_(3ax) ^(NeuNAc) _(UR1) 1.782 H_(3ax) ^(NeuNAc) _(UR2) 1.726 H_(3ax)^(NeuNAc) _(UR3) 1.705 H_(3ax) ^(NeuNAc) _(UR4) 1.705 The ¹H NMR spectraconfirmed the molecular structure, identity and integrity of saccharidechains, and show the DP values of the samples: DP_(MenW135) = 3;DP_(MenY) = 4.

Thus the Q-Sepharose column was able to resolve oligosaccharides by DP,and the ESI and NMR analyses confirm that the oligosaccharide standardsare: MenW135 DP3; and MenY DP4. These standards were analysed by theprocesses of the invention.

Chromatographic analysis of DP

Oligosaccharide samples were adjusted to contain 0.5 mg/ml sialic acidin 100 μl. These samples were treated with 100 μl NaBH₄ solution 40 mMin NaOH 10 mM. Samples were heated at 37° C. for 2 hours in a closedscrew-cap test tube. To stop the reaction samples were then treated with10 μl ammonium acetate 5M pH 6.0 and maintained at room temperature for30 minutes. 200 μl methanol was added and samples were then dried on aSpeed Vac concentrator fitted with a refrigerated condensation trap(Savant SC110) under vacuum for 1 hour.

Samples were reconstituted with 100 μl Milli-Q water and 100 μl TFA 4M(final concentration: 2M) and heated at 100° C. for 90 minutes.Hydrolysates were then dried on a Speed vac concentrator fitted with tworefrigerated condensation trap (Savant SC 110).

For HPAEC-PAD analysis, samples were dissolved in 1.0 ml Milli-Qdegassed water and then filtered (0.22 μm). Analysis of the hydrolysedproducts was performed on the same Dionex system, but using a CarbopacPA1 column (4×250 mm) with a Borate Trap guard column. This column andguard are better suited to monosaccharide analysis that the PA100 andAS11 columns. Isocratic elution with sodium acetate 50 mM+sodiumhydroxide 20 mM was used in some experiments, and other experiments usedgradient elution was used with the following eluents: A) sodium acetate100 mM+sodium hydroxide 20 mM and B) sodium hydroxide 20 mM and with agradient from 10% to 70% of A (curve 7). Eluates were analysed asdescribed above.

This apparatus can distinguish sialic acid from sialitol, and can givequantitative results for each. The ratio of total (i.e. reduced andnon-reduced) sialic acid to reduced sialic acid was used to calculatethe DP of the starting oligosaccharides.

For initial testing, a standard solution of pure sialic acid (Sigma,Steinheim, Germany) was prepared at 2.0 μg/ml and subjected to NaBH₄reduction as described above. Samples were analysed at various timepoints by HPAEC with isocratic elution. Treatment with 0.04M NaBH₄ for 2hours at 37° C. was found to give complete reduction of sialic acid inthe standard. FIG. 10 shows a time zero sample, and FIG. 11 shows the 2hour sample, with retention time decreasing from 8.5 to 4.7-5.0.

The double peak in FIG. 11 is due to unresolved diastereoisomers ofsialitol [14]. Treatment of the sialic acid standard with 2M TFA for 90minutes at 100° C. was able to remove the double peak (FIG. 12), thusgiving a single peak for sialitol quantification. TFA hydrolysis isknown to be suitable for saccharide cleavage, in terms of bothefficiency and ease of removal [82-84], and has been used for analysisof several saccharide vaccines [82]. Thus the use of TFA for acidhydrolysis of saccharides has a triple purpose—efficient hydrolysis,efficient removal, and removal of peak splitting for sialitol.

An oligosaccharide sample of MenY, with an expected DP ranging between 3and 5, was treated as described, but isocratic conditions forchromatographic elution did not give good separation of the sialitolpeak. An elution gradient was therefore used instead, which gave thechromatogram shown in FIG. 13. Sialitol and sialic acid had retentiontimes of 19.3 and 29.8 minutes, respectively. A sample of MenW135oligosaccharide with an expected DP ranging between 3 and 5 was analysedwith the same method (FIG. 20). Sialitol is seen at 19.33 minutes andsialic acid is seen at 29.77.

To facilitate quantitative analysis of the FIG. 13 and FIG. 20chromatograms, standard curves based on known sialic acid and sialitolconcentrations were made. The standard curve used 0.5, 1.0, 2.0, 4.0 and6.0 μg/ml sialic acid or sialitol. The linear response of the detectoris shown in FIG. 14.

By comparison to these standard curves, amounts of sialitol and sialicacid could be quantified in the MenW135 and MenY samples. For example,the area under the 19.33 minutes peak in the MenW135 elution (FIG. 20)was calculated to be 44914068. By reference to the standard curve, thesialitol concentration was calculated to be 7.96 μg/ml. In a duplicateexperiment a concentration of 8.05 μg/ml was seen. The two analyses givea mean value of 8.005 μg/ml. As the samples were diluted 10× prior toanalysis then the original mean sialitol concentration was 80.05 μg/ml.Colorimetric detection of total sialic acid gave a concentration of241.09 μg/ml. Taking account of the slight mass difference betweensialic acid and sialitol, these concentrations were converted to molarconcentrations (in fact, the mass difference is so slight that anexcellent approximation is achieved without converting to moles) and themolar ratio was calculated as 3 (i.e. DP=3). Ratios calculated fromresults of two different duplicate analyses were as follows:

Sample Sialitol (μg/ml) Sialic acid (μg/ml) DP Oligo-MenW 80.5 241.093.0 Oligo-MenW 79.6 241.09 3.0 Oligo-MenY 62.7 303.64 4.8 Oligo-MenY68.9 303.64 4.4

Repeatability of the sialitol measurement method was evaluated byanalysing a single MenW135 sample for 4 replicates using three differentpre-column borate traps. Results were as follows:

Borate Sialitol Sialic acid trap (μg/ml) (μg/ml)* DP Average Std Dev CV% 1 342.5 6484.2 18.9 18.7 0.2 0.9 345.7 6484.2 18.8 346.3 6484.2 18.7350.2 6484.2 18.5 2 335.7 6484.2 19.3 19.2 0.1 0.5 339.5 6484.2 19.1338.3 6484.2 19.2 336.0 6484.2 19.3 3 328.0 6484.2 19.8 19.7 0.4 1.9335.5 6484.2 19.3 334.3 6484.2 19.4 321.8 6484.2 20.2 *Measuredseparately, duplicate analysis

The results thus show a good repeatability (CV %<2%).

A 10 mg/ml solution of full-length MenY or MenW135 CPS in 0.01 M aceticacid was heated to 70-80° C. During hydrolysis, samples were taken foraverage DP determination to track the progress of the reaction for up to6 hours. Samples were rapidly frozen and maintained at −20° C. prior toduplicate analysis using an IonPac AS11 column. Results were as follows:

MenW135 MenY Sialic Sialic Time Sialitol acid Average Sialitol acidAverage (hours) (μg/ml) (μg/ml) DP (μg/ml) (μg/ml) DP 0.5 45.40 5338.4117.6 61.56 5903.0 95.9 1.0 74.12 5351.5 72.2 94.84 5803.6 61.2 1.5125.63 5519.9 43.9 125.87 5764.0 45.8 2.0 139.49 5620.2 40.3 170.925790.0 33.9 2.5 195.40 5611.8 28.7 218.13 5808.4 26.6 3.0 235.55 5555.723.6 237.17 6294.1 26.5 3.5 256.90 5762.3 22.4 267.25 6196.6 23.2 4.0270.83 5491.2 20.3 286.79 6188.8 21.6 4.5 296.09 5586.8 18.9 376.956715.1 17.8 5.0 336.06 5678.7 16.9 440.85 6314.1 14.3 5.5 384.09 5560.514.5 506.29 6400.3 12.6 6.0 423.32 5629.8 13.3 — — —

Thus the DP measurements show a gradual decrease in DP over time (FIG.15). Confirmation of the depolymerisation was obtained with IonPac AS11chromatographic analysis that shows how low molecular weightoligosaccharide content increases with time during acid hydrolysis, asshown in FIGS. 16 (MenW315) & 17 (MenY), where 16A/17A is time zero and16B/17B is after 6 hours.

Serogroup W135 sample

Capsular saccharide from serogroup W135 was prepared. Duringpreparation, its DP was measured using the methods disclosed herein. Achromatogram for a 100-fold dilution of this material is shown in FIG.21B, with a 6 μg/ml standard being shown in FIG. 21A. Elution in bothcases used 100 mM sodium acetate plus 20 mM NaOH, and is shown risinglinearly from 10% elution buffer to 30% elution buffer over 30 minutes,with a final jump to 100% elution buffer for 10 minutes.

Sialitol in the standard was shown to elute at 16.383 minutes; in thesample there was a peak at 16.350 minutes.

Analysis characteristics from two separate analyses of the same sample,and five standard samples, are shown below, together with the calculatedconcentration of sialitol, were as follows:

Retention Time Area Height Amount Sample Name (min) (nC * min) (nC)(μg/ml) std1 16.700 0.8446 1.80 0.5023 std2 16.584 1.6986 3.50 1.0103std3 16.134 3.4018 6.33 2.0234 std4 16.567 6.8201 14.27 4.0566 std516.384 10.0077 19.34 5.9526 W135(a) 16.350 5.4644 11.35 3.2502 W135(b)16.600 5.7620 12.46 3.4272 Based on the mean of analyses (a) and (b),there was 3.339 μg/ml sialitol in the sample. Adjusting for the initialdilution, the original sialitol concentration was 333.9 μg/ml. Totalsialic acid was measured as 5732.6 μg/ml, giving a DP value of 17.2.Serogroup C analysis

Purified MenC DP5 oligosaccharide was obtained as previously described[5]. ESI MS analysis was performed in the positive ion mode. The massspectrum is shown in FIG. 18, where three main group of ions areevident. Detailed inspection of the peaks with the highest intensity(856-941 m/z) indicated that they correspond to doubly chargedpentasaccharides while peaks ranging from 1733-1826 m/z correspond tosingle charged pentasaccharides, both differing for the number ofO-acetyl substituents and of sodium as counter ions.

The DP of MenC DP5 oligosaccharides was determined with thechromatographic methods of the invention, and a chromatogram of theanalysis is shown in FIG. 19. Four separate analyses of theoligosaccharides were prepared, and results were as follows:

Sialitol Sialic acid (μg/ml) (μg/ml) DP Average Std Dev CV % 160.7 845.45.3 5.0 0.2 3.1 170.3 845.4 5.0 169.1 845.4 5.0 172.2 845.4 4.9Conclusions

The molecular size of saccharides has been an important issue in thedesign of previous conjugate vaccines [6,7], with intermediatechain-length oligosaccharides showing better immunogenicity.Preparation, isolation and characterisation of intermediate MenW135 andMenY oligosaccharides has been shown, with acid hydrolysis driving theinitial depolymersiation. of the capsular polysaccharide [79]. Theinvention offers a new chromatographic method for determining the degreeof polymerisation of these oligosaccharides, and the method shows goodprecision and accuracy, confirmed by NMR and ESI-MS analysis. Moreover,the method can be used also for determining DP of oligosaccharides fromserogroup C.

Additional experimental details can be found in reference 85.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

References (The Contents of which are Hereby Incorporated by Reference)

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We claim:
 1. A method for preparing a vaccine composition, comprisingthe steps of: (a) providing a capsular saccharide whose degree ofpolymerization (DP) has been measured by a process comprising the stepsof: (i) reducing the terminal sialic acid monosaccharide subunit to givea reduced sialic acid monosaccharide subunit; (ii) hydrolyzing thesaccharide to give a hydrolysate containing monosaccharide subunits,wherein the hydrolysis proceeds to completion to yield onlymonosaccharide subunits; and (iii) determining the ratio of total sialicacid to reduced sialic acid in the hydrolysate, wherein the capsularsaccharide comprises sialic acid monosaccharide subunits that are linkedα 2→9 and has a terminal sialic acid monosaccharide subunit; (b)conjugating the DP-analyzed capsular saccharide to one or more carrierproteins; (c) analyzing the bulk vaccine comprising the conjugatedcapsular saccharide for pH and/or other properties; and, if the resultsfrom step (c) indicate that the bulk vaccine is acceptable for clinicaluse, (d) preparing and packaging the vaccine for human use from the bulkvaccine.
 2. The method of claim 1, wherein the terminal sialic acidresidue is reduced by incubating the saccharide with a reducing agent.3. The method of claim 2, wherein the reducing agent is sodiumborohydride.
 4. The method of claim 1, wherein acid hydrolysis is used.5. The method of claim 2, wherein acid hydrolysis is used.
 6. The methodof claim 3, wherein acid hydrolysis is used.
 7. The method of claim 1,where the capsular saccharide is a population of different-sizedcapsular saccharides, and the process provides the average degree ofpolymerization of the capsular saccharides.
 8. The method of claim 2,where the capsular saccharide is a population of different-sizedcapsular saccharides, and the process provides the average degree ofpolymerization of the capsular saccharides.
 9. The method of claim 3,where the capsular saccharide is a population of different-sizedcapsular saccharides, and the process provides the average degree ofpolymerization of the capsular saccharides.
 10. The method of claim 4,where the capsular saccharide is a population of different-sizedcapsular saccharides, and the process provides the average degree ofpolymerization of the capsular saccharides.
 11. The method of claim 7,where the capsular saccharide is a population of different-sizedcapsular saccharides, and the process provides the average degree ofpolymerization of the capsular saccharides.
 12. The method of claim 1wherein step (ii) of the process further comprises startingdepolymerization of the capsular saccharide; and, at one or more timepoints thereafter prior to complete hydrolysis, measuring DP of thecapsular saccharide by determining the ratio of total sialic acid toreduced sialic acid in the hydrolysate.
 13. The method of claim 2wherein step (ii) of the process further comprises startingdepolymerization of the capsular saccharide; and, at one or more timepoints thereafter prior to complete hydrolysis, measuring DP of thecapsular saccharide by determining the ratio of total sialic acid toreduced sialic acid in the hydrolysate.
 14. The method of claim 3wherein step (ii) of the process further comprises startingdepolymerization of the capsular saccharide; and, at one or more timepoints thereafter prior to complete hydrolysis, measuring DP of thecapsular saccharide by determining the ratio of total sialic acid toreduced sialic acid in the hydrolysate.
 15. The method of claim 10wherein step (ii) of the process further comprises startingdepolymerization of the capsular saccharide; and, at one or more timepoints thereafter prior to complete hydrolysis, measuring DP of thecapsular saccharide by determining the ratio of total sialic acid toreduced sialic acid in the hydrolysate.
 16. The method of claim 11wherein step (ii) of the process further comprises startingdepolymerization of the capsular saccharide; and, at one or more timepoints thereafter prior to complete hydrolysis, measuring DP of thecapsular saccharide by determining the ratio of total sialic acid toreduced sialic acid in the hydrolysate.
 17. The method of claim 1wherein the capsular saccharide is from meningococcal serogroup W135 orserogroup Y.
 18. The method of claim 3 wherein the capsular saccharideis from meningococcal serogroup W135 or serogroup Y.
 19. The method ofclaim 7 wherein the capsular saccharide is from meningococcal serogroupW135 or serogroup Y.